Cheilolejeunea zhui (Lejeuneaceae, Marchantiophyta), a new species with moniliate ocelli from Guangxi, China

Abstract A new ocellate liverwort species, Cheilolejeunea zhui (Lejeuneaceae), is described from Guangxi, China. The new species is similar to the neotropical C. urubuensis in having moniliate ocelli in the leaf lobes and in general appearances but differs in having obliquely spreading leaves, obtuse to subacute leaf apex, thin‐walled leaf cells with distinct trigones, shallowly bifid female bracteole apex, and numerous ocelli in its perianths. Molecular phylogeny of data from three regions (nrITS, trnL–F, and trnG) confirmed the systematic position of this new species to be sister to C. urubuensis, well apart from the remaining members of the genus. Based on morphological and molecular evidence, Cheilolejeunea sect. Moniliocella sect. nov. is proposed to accommodate C. urubuensis and C. zhui. The discovery of C. zhui represents the fourth known species in Cheilolejeunea with linearly arranged ocelli.

Interestingly, almost all of these recent records are representatives of oligotypic genera.
During a recent expedition to northern Guangxi, a small population of an unfamiliar species of Cheilolejeunea (Spruce) Steph.
was encountered (Figure 1). Cheilolejeunea is one of the largest and most diverse genera of Lejeuneaceae, with a pantropical distribution (Ye et al., 2015). Recent molecular phylogenetic research on Lejeuneaceae has settled a broader concept of the genus (Schäfer-Verwimp et al., 2014;Wilson et al., 2007;Ye et al., 2015), which shown that several related genera of Lejeuneaceae, such as  (Gradstein & Reiner-Drehwald, 2017;Ye et al., 2015). Therefore, the main key diagnostic features of Cheilolejeunea are, the distal hyaline papilla on the leaf lobule, and the leaf cells with few large, coarsely granular (rarely finely granular) oil bodies per cell. The number of accepted species in the genus has reached over 200 (Söderström et al., 2016).
However, since a worldwide monograph of Cheilolejeunea is still lacking, the actual number of species in the genus may be reduced to an estimated 80-100 species (Bastos & Gradstein, 2020;Ye et al., 2015).
This unusual species that we have encountered along a riverside trail in Guangxi, differs from all described members of Cheilolejeunea by having a single row of moniliform ocelli in its leaf lobe, and numerous ocelli in perianth. In this paper, we describe this distinctive species as Cheilolejeunea zhui, a new species supported by both morphological and phylogenetic evidence.

| Morphological observations
The morphological and anatomical characters were examined and photographed using an Olympus SZX7 stereomicroscope and an Olympus BX43 light microscope equipped with a digital camera (Mshot MH125). Field images were taken with a digital camera (Canon G16). Drawing of the voucher specimens was produced using an Olympus BX43 microscope equipped with a drawing tube.

| Taxon sampling, DNA extraction, PCR amplification, sequencing, and alignment
We included one freshly collected sample of Cheilolejeunea zhui and one sample of C. urubuensis (Zartman & Wei (Colombia, Campos M6-P) in our molecular phylogenetic study.
Sequences of representatives in the subfamily Ptychanthoideae were selected as an outgroup following the phylogenetic results of Wilson et al. (2007). Sequences of representative species from various subtribes of subfamily Lejeuneoideae were also obtained from GenBank (http://www.ncbi.nlm.nih.gov/genba nk/), namely Steph., and Pycnolejeunea (Spruce) Schiffn. Sixty-one samples from Cheilolejeunea representing 50 species and nine out of ten sections of the genus were also included in the dataset (Bastos & Gradstein, 2020;Ye et al., 2015).
Voucher information and GenBank accession numbers are presented in Table 1.
The DNA extraction, PCR, and DNA sequencing methods follow Ye and Zhu (2018). Three molecular genetic makers, nrITS F I G U R E 1 A lowland valley at Guangxi, where Cheilolejeunea zhui was found on tree bases and roots along a riverside trail. TA B L E 1 List of specimen vouchers and GenBank accession numbers used in this study. (ITS1-5.8S-ITS2), trnL-F, and trnG were sequenced. Primer references for these regions are given in Ye et al. (2015).
Bidirectional sequences were assembled using PhyDE v.1 (Müller et al., 2010) and then aligned manually in PhyDE v.1 with other sequences of 71 selected Lejeuneaceae species. Ambiguous hotspot positions were excluded prior to molecular phylogenetic analyses; gaps were coded as missing data.

| Phylogenetic analyses
Preliminary phylogenetic analyses of the dataset of each marker revealed topological congruence and based on this result they were combined for subsequent analyses. PRAP2 (Müller, 2007) applying the default settings was employed for generating command files using the parsimony ratchet (Nixon, 1999). Maximum Parsimony (MP) analyses were then performed with these command files executed in PAUP* 4.0b10 (Swofford, 2000). Heuristic bootstrap searches under parsimony were performed with 10,000 replicates. with gamma-distributed rate variation (+G) and invariable sites (+I) for the nrITS region. MrModeltest v2.4 (Nylander, 2004) was used to establish the best-fit model for Bayesian inference (BI), and identified a General Time Reversible (GTR) model, with gammadistributed rate variation (+G) and invariable sites (+I) as the best-fit model for the trnL-F region and nrITS region, and GTR+ G for the trnG region.
The ML trees were generated using the program GARLI version 2.01 (Zwickl, 2006). Best ML trees were found with partitioned analysis based on five independent searches. Bootstrap support (BS) was estimated based on 1070 bootstrap replicates. A partitioning scheme by molecular marker and the same nucleotide substitution models as described above were implemented.
Bayesian inference (BI) was undertaken using MrBayes 3.2.7a (Huelsenbeck & Ronquist, 2001) on the CIPRES Science Gateway  Accession numbers of newly generated sequences are in bold.

TA B L E 1 (Continued)
runs were combined for the final inference of posterior probabilities (PP) of both trees and model parameters after discarding the burn-in.

| Morphological observations
Morphological observations in the field showed that fresh plants of

| Phylogenetic analyses
The

| DISCUSS ION
The present species clearly belongs to Cheilolejeunea in having hya- Ocelli, the specialized oil cells filled by a single oil body, are widespread in the family Lejeuneaceae, especially in the subfamily Lejeuneoideae (He & Piippo, 1999). Ocelli are regarded as taxonomically important at generic and specific levels and have evolved more than once in Lejeuneoideae (Dong et al., 2013).
Ocelli in other ocellate Cheilolejeunea species are arranged in linear form (He & Piippo, 1999), and occur continuously and longitudinally from leaf base to at least the middle of the leaf lobe. In